This invention relates to methods of affecting retinal cell function.
The invention relates to prophylactic or affirmative treatment of diseases and disorders of retina and associated tissues of the eye by administering polypeptides found in vertebrate species, which polypeptides are growth, differentiation and survival factors for several cell types. Normal function of retinal cells including survival, proliferation, differentiation, and maintenance is dependent upon the controlled expression of a variety of peptide growth factors. Some of these factors can be produced by neuronal cells and by other cells of the retina, which provide a signal to regulate retinal cell function.
Anatomy and Function of the Retina
The retina is that component of the visual system which senses light and transmits impulses via the optic nerve to the visual cortex where the signals are deciphered and interpreted as images. The retina is comprised of a series of layers and cell types as illustrated in FIG. 1.
The basic function of the retina is to transduce the visual image into a pattern of electrical potential changes that can be processed by the visual centers in the brain. The changes in electrical potentials in the retinal cells are then relayed to the brain. The structure of the retina reflects these functions (FIG. 1). The cells of the retina are arrayed in three layers: (1) the outer nuclear layer, which contains the photoreceptor cells; (2) the inner nuclear layer, which contains the cell nuclei of most of the retinal interneurons and glia; and (3) the ganglion cell layer, which contains the cell bodies, of the cells that relay the visual information to the brain via the optic nerve. In addition to these nuclear layers, there are three other distinct layers in the retina. The outermost layer is composed of the outer segments of the photoreceptor cells; this is where the actual process of light-to-electrical signal transduction take place. The outer plexiform layer lies between the outer and inner nuclear layers. It is made up of synapses between the terminals of the photoreceptors and the dendrites of the retinal interneurons of the inner nuclear layer. The inner plexiform layer lies between the inner nuclear layer and the ganglion cell layer. This layer is where the interneurons of the inner nuclear layer synapse with the retinal ganglion cell dendrites.
The retina is composed of five classes of neurons, and two classes of supporting cells (Principles of Neural Science, 3rd ed., Ed. by E. R. Kandel, J. H. Schwartz, and T. M. Jessell, Elsevier, New York, N.Y. 1991). Of the neuronal types, the receptor cells are the cells that transduce light into electrical signals. Receptor cells are of two subtypes: conesxe2x80x94which mediate form and color perception in daylight, and rodsxe2x80x94which mediate form perception in dim light. Ganglion cells of the retina project axons into the brain via the optic nerve and are the output cells of the retina. The remaining neuronal types are interneurons that modulate retinal output: bipolar cells connect receptor cells to ganglion cells; horizontal cells mediate lateral interactions between receptors and bipolar cells; and amacrine cells mediate lateral interactions between bipolar cells and ganglion cells. The supporting cell types are the glial cells of the retina, Mxc3xcller cells, and the pigment epithelium cells. The latter cell type plays an important role in the maintenance of receptor cells.
The basic flow of information through the retina is as follows (Refer to FIG. 1): (1) light passes through the cells of the retina and is absorbed by the outer segments of the photoreceptor cells; (2) the photons are transduced into potential changes in the photoreceptor cells; (3) this change in potential is relayed to one type of retinal interneuron in the inner nuclear layer, the bipolar cell, via synapses in the outer plexiform layer; (4) the bipolar cells relay the electrical potential changes to the ganglion cells through their synapses in the inner plexiform layer; and (5) the ganglion cells convert the potential changes into action potentials that are sent along the optic nerve to the brain. This process results in a pattern of action potentials in the optic nerves that reflects the pattern of light and dark in the visual world. Some initial processing of the visual information takes place in the retina before it is relayed to the other visual areas in the brain.
Proper development and maintenance of the retina is necessary for sustaining normal vision. Degeneration of components of the retina can lead to partial or total blindness.
Peptide Growth Factors
The development and physiology of multicellular organisms requires multiple modes of intercellular communication. Such communication may be systemic, as in the case of hormones delivered via the bloodstream, or can be highly localized. In the latter case two modes are commonly recognized: synaptic signaling from neurons, and paracrine signaling from adjacent or nearby cells (Molecular Biology of the Cell, Alberts et al., 2nd ed. Garland Publishing, New York, N.Y. 1989). A function of such signaling is to coordinate cell survival, proliferation, differentiation, and/or metabolic activity. The molecules that serve as transmitted signals vary in their chemical composition; one group of molecules are proteins, the peptide growth factors. Peptide growth factors act upon cells by binding to cell surface receptors. These receptors are coupled to intracellular signal transduction pathways that give rise to the above described activities when activated by growth factor binding. The genesis and differentiation of the varied retinal cell types and the generation of distinct layers in the retina from progenitor cells of the optic cup are the result of developmental events that are mediated by intercellular communication involving peptide growth factors.
Peptide Growth Factors in the Retina
The roles of growth factors in the development and maintenance of the retina have been studied in cell culture, by molecular analysis of the expressed growth factors and their receptors, and in animal models of disease or injury.
As an example of in vitro studies, explants and partially-dissociated chick retinal pigmented epithelium (RPE) can trans-differentiate into neural retina in the presence of bFGF (Coulombre and Coulombre, Dev. Biol. 12:79, 1965). Proliferation of dissociated RPE cells is stimulated by aFGF, bFGF, EGF, PDGF, IGF, and insulin; and it is inhibited by TGFb (Sternfeld et al., Curr. Eye Res. 8: 1029, 1989; Leschey et al., Invest. Ophthalmol. Vis. Sci. 31: 839, 1990; Song and Lui, J. Cell Physiol. 143:196, 1990). Cultured RPE cells are induced by cytokines to release nitric oxide, which is cytotoxicxe2x80x94and the induction can be blocked by FGF (Goureau et al., Biochem. Biophys Res. Comm. 186:854, 1992; op. cit., 198: 120, 1994). Further, retinal explants from the rd mouse are rescued from cell death by combined treatment with NGF and bFGF (Caffe et al., Curr. Eye Res. 12:719, 1993).
The presence of growth factor receptors in retinal cells has been demonstrated by a variety of molecular analytical techniques, including immunostaining, in situ hybridization and tissue binding using radio-labeled ligands. Cells in the RPE express FGF receptors (Malecaze et al., J. Cell Physiol. 154: .1105, 1993). Ganglion cells and Mxc3xcller cells express receptors for BDNF, CNTF, FGF, trkA and trkB (Jelsma et al., J. Neurobiol. 24:1207, 1993; Takahashi et al., Neurosci. Lett. 151:174, 1993; Carmignoto et al., Exp. Neurol. 111:302 191; reviewed in Steinberg, Curr. Opin. Neurobiol. 4:515, 1994). Mxc3xcller cells also express PDGF receptors (Mudhar et al., Development 118: 539, 1993). Receptors for IGF are detected on photoreceptor cells (Waldbillig et al., Exp. Eye Res. 47:587 1988; Ocrant et al., Exp. Eye Res. 52:581, 1991), and depending on the species and developmental stage that are analyzed receptors for bFDF have been localized on several cell types, including retinal ganglion cells (Sternfeld et al., Curr. Eye Res. 8:1029, 1992; Schweigerer et al., Biochem Biophys. Res. Comm. 143:934, 1987).
Studies on retinal ganglion cell survival in vivo in animal models of optic nerve axotomy and retinal ischemia have demonstrated effects due to FGF (Sievers et al., Neurosci. Lett. 76:157, 1987), NGF (Carmignoto et al., J. Neurosci. 2:1263, 1989), CNTF (Mey and Thanos, Brain Res. 602:304, 1993), BDNF (Mansour-Robaey et al., PNAS USA 91:1632, 1994; Mey and Thanos, Brain Res. 602:304, 1993), NT4/5 (Cohen et al., J. Neurobiol. 25:953, 1994) and bFGF (Ferguson et al., J. Neurosci. 10:2176, 1990). Some undesirable retinal complications, including macrophage proliferation, inflammation, disorganization of retinal structure and angiogenesis are associated with treatment of the retina with several of the above factors.
Neuregulins
A recently described family of growth factors, the neuregulins (reviewed, by Mudge, Curr. Biol. 3:361, 1993; Peles and Yarden, Bioessays 15:815, 1993), are synthesized by neurons (Marchionni et al. Nature 362:313, 1993) and by mesenchymal cells from several parenchymal organs (Meyer and Birchmeier, PNAS 91:1064, 1994). The neuregulins and related factors that bind p185erbB2 have been purified, cloned and expressed (Benveniste et al. PNAS, 82:3930, 1985; Kimura et al., Nature 348:257, 1990; Davis and Stroobant, J. Cell Biol. 110:1353, 1990; Wen et al., Cell 69:559, 1992; Yarden and Ullrich, Ann. Rev. Biochem. 57:443, 1988; Dobashi et al., Proc. Natl. Acad. Sci. 88:8582, 1991; Lupu et al., Proc. Natl. Acad. Sci. 89:2287, 1992; Wen et al., Mol. Cell. Biol. 14:1909, 1994). Recombinant neuregulins have been shown to be mitogenic for peripheral glia (Marchionni et al., Nature 362:313, 1993) and have been shown to influence the formation of the neuromuscular junction (Falls et al., Cell 72:801, 1993; Jo et al., Nature 373: 158, 1995; Chu et al., Cell 14: 329, 1995).
The neuregulin gene consists of at least thirteen exons. The neuregulin transcripts are alternatively spliced and these encode many distinct peptide growth factors, which are referred to as the neuregulins (Marchionni et al., Nature 362:313, 1993). DNA sequence comparisons revealed that neu differentiation factor (NDF) (Wen et al., Cell 69:559, 1992) and heregulins (Holmes et al., Science 256:1205, 1992), which were purified as ligands of the p185erbB2 (also known as neu or HER2) receptor tyrosine kinase, also are splicing variants of the neuregulin gene. The acetylcholine receptor inducing activity (ARIA) also is a product of the neuregulin gene (Falls et al., Cell 72:801, 1993). Common structural features of the neuregulins are the presence of a single immunoglobulin-like (Ig) fold and a single epidermal growth factor-like (EGF) domain.
The sites of neuregulin gene expression have been characterized by use of nucleic acid probes to analyze RNA samples by a variety of methods, such as Northern blotting, RNase protection, or in situ hybridization. Transcripts have been detected in the nervous system and in a variety of other tissues (Holmes et al., Science 256:1205, 1992 Wen et al., Cell 69:559, 1992; Meyer and Birchmeier, PNAS 91:1064, 1994). Sites of gene expression have been localized in the brain and spinal chord and in other tissues. (Orr-Urteger et al., PNAS 90:1.867, 1993; Falls et al., Cell 72:801, 1993; Marchionni et al., Nature 362:313, 1993; Meyer and Birchmeier, PNAS 91:1064, 1994; Chen et al., J. Comp. Neurol. 349; 389, 1994; Corfas et al., Neuron 14:103, 1995). Specifically in the retinal neurepithelium, expression of neuregulin transcripts has been detected at embryonic day 18 in rat (Meyer and Birchmeier, PNAS 91:1064, 1994).
Although a large number of neuregulins may be produced by alternative splicing, they can be broadly sorted into the putative membrane-bound and the soluble isoforms. The former contains a putative trans-membrane domain and may be presented at the cell surface. Membrane-anchored peptide growth factors may mediate cell-cell interactions through cell-adhesion or juxtacrine mechanisms (reviewed by Massaguxc3xa9 and Pandiella, Ann. Rev. Biochem. 62:515, 1993). Alternatively, the putative membrane-bound isoforms may be cleaved from the cell surface and function as soluble proteins (Wen et al., Cell 69.559, 1992; Falls et al., Cell 72:801, 1993). The soluble neuregulin isoforms contain sequence corresponding to the extracellular domains of the putative membrane-bound isoforms, but terminate before the transmembrane domain. These neuregulin isoforms may be secreted, and hence could affect cells at a distance; or they may be present in the cytoplasm, but could be released upon cellular injury. In the latter case, neuregulins may function as injury factors, as has been postulated for the ciliary neurotrophic factor (Stxc3x6ckli et al., Nature 342:920, 1989). Any one of these modes of action of the neuregulins may occur in the retina.
Cellular targets of peptide growth factors are those which bear receptors for the factor(s) and those that are shown to respond in a bioassay either in vitro or in vivo. Based on studies demonstrating phosphorylation on tyrosine residues or cross-linking experiments, neuregulins are candidate ligands for the receptor tyrosine kinases p185erbB2 (or HER-2 in human), p185erbB3 (HER-3 in human), p185erbB4 (or HER-4 in human) or related members of the EGFR gene family. Collectively, these receptors can be referred to as erbB receptors. Though the precise ligand-receptor relationship of each neuregulin protein with each member of the EGFR family is yet to be clarified, several lines of evidence suggest that binding of ligands is mediated by either erbB3 and erbB4, but signaling occurs through either erbB2, erbB4 and heterodimers of the various subunits (e.g., Carraway and Cantley, Cell 78:5, 1994). These receptors are known to be present on Schwann cells and muscle cells (Jo et al., Nature 373: 158, 1995), and other neuregulin targets, such as cell lines derived from various tumor tissues, such as breast and gastric epithelia. Sites of expression of the HER-4 gene have been localized by in situ hybridization to several regions of the brain, including: hippocampus, dentate gyrus, neo cortex, medial habenula, reticular nucleus of the thalamus, and the amygdala (Lai and Lemke, Neuron 6:691, 1991). The distribution of the HER-4 receptor has not been studied by methods that allow detection of the protein or the activated receptor tyrosine kinase in vivo or in cultures of primary cells. The expression pattern of erbB2, erbB3 and erbB4 in the retina has not been described.
Neuregulins have been shown to have a variety of biological activities depending on the cell type being studied. Several neuregulins, including native bovine GGFI, II and III and recombinant human GGF2 (rhGGF2) are mitogenic for Schwann cells (Marchionni et al., Nature 362:313, 1993), as is heregulin B1 (Levi et al, J Neurosci. 15:1329, 1995). On human muscle culture, rhGGF2 has a potent trophic effect on 7 myotubes (Sklar et al., U.S. patent application Ser. No. 08/059, 022), filed May 6, 1993, now abandoned. The differentiation response to rhGGF2 also includes induction of acetylcholine receptors in cultured myotubes (Jo et al., Nature 373: 158, 1995). This activity is associated with other forms of neuregulin, including ARIA (Falls et al., Cell 72:801, 1993) and heregulin B1 (Chu et al., Neuron 14:329, 1995), as well as with rhGGF2. Further, ARIA has been shown to induce synthesis of voltage-gated sodium channels in chick skeletal muscle (Corfas and Fischbach, J. Neurosci. 13:2118, 1993). Glial growth factor (GGF), and more specifically rhGGF2, can restrict neural crest stem cells to differentiate into glial cells in vitro (Shah et al., Cell 77:349, 1994). Activities of neuregulin on retinal cells have not been described. In summary, there are examples of neuregulin proteins affecting proliferation, survival and differentiation of target cells.
Pharmaceutical Need for Treating Disorders of the Eye
A variety of retinal diseases and related disorders are known that produce impaired vision and in some cases progress to total blindness. These disorders of the eye include, but are not necessarily limited to: various retinopathies, such as hypertensive retinopathy, diabetic retinopathy and occlusive retinopathy; also injuries and disorders resulting in retinal degeneration, such as retinal tearing and detachment and inherited diseases, such as retinitis pigmentosa; also age-related macular degeneration; diseases of the optic nerve; glaucoma and retinal ischemia.
Diabetic Retinopathy is the leading cause of blindness in patients 25-74 years. It is responsible for 12,000-24,000 new cases of blindness per year in the United States. Of the 6 million diabetics in the US 50% show detectable retinopathy after 7 years of diabetes. Age-related macular degeneration (ARMD) is estimated to be present in over 9% of the population 52 years and older and in 33% of the population 75 years and older. Glaucoma is associated with chronically high intraocular pressure and approximately 2 million people in the US are currently being treated. In the US approximately 100,000 people are blinded each year by glaucoma.
There is precedent for the use of growth factors that have been shown to be active on retinal cultures in the treatment of retinal degenerative diseases. FGF supports the survival of photoreceptor cells in culture and has been injected into the extracellular space surrounding the rods and cones or into the vitreous body to rescue the photoreceptors in rats which have degeneration as a result of light damage or because of an inherited disease (LaVail et al, PNAS 89: 11249, 1992, Faktorovich et al J. NeuroSci 12: 3554, 1992). Similarly TGFb2 has been used for the treatment of Macular holes in humans. The TGFb used was derived from bovine sources and was administered by directly infusing the factor into the area of the macular hole (Glaser et al., Opthalmol. 99: 1162, 1992).
Currently, there are limited options for therapy for the promotion of retinal cell function, including survival, proliferation, differentiation, growth and changes in gene activity and metabolic activity. Such a therapy would be useful for treatment of a variety of eye disorders resulting in loss of sight.
In general, the present invention provides methods for promoting the function of retinal cells using neuregulins. A novel aspect of the invention involves the use of neuregulins as growth factors to promote survival of retinal cells. Treating of the retinal cells to provide these effects may, be achieved by contacting retinal cells with a polypeptide described herein. The treatments may be provided to slow or halt net cell loss or to increase the amount or quality of retinal tissue present in the vertebrate.
Neuregulins are a family of protein factors heretofore described as glial growth factors, acetylcholine receptor inducing activity (ARIA), heregulins, neu differentiation factor, which are encoded by one gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to and activation of erbB2 (neu) and closely related receptors erbB3 and erbB4. The invention provides methods for using all of the known products of the neuregulin gene, as well as, other not yet discovered splicing variants of the neuregulin gene. Thus, the above factors, regulatory compounds that induce synthesis of these factors, and small molecules which mimic the effect of these factors by binding to the receptors on retinal tissues or by stimulating through other means the second messenger systems activated by the ligand-receptor complex are all extremely useful as prophylactic and affirmative therapies for retinal tissue diseases and related disorders of the eye.
The survival of retinal cells as used herein refers to the prevention of loss of retinal cells by necrosis or apoptosis or the prevention of other mechanisms of retinal loss. Survival as used herein indicates a decrease in the rate of cell death of at least 10%, more preferably by at least 50%, and most preferably by at least 100% relative to an untreated control. The rate of survival may be measured by counting cells stainable with a dye specific for dead cells (such as propidium iodide) in culture.
Methods for treatment of diseases or disorders using the polypeptides or other compounds described herein are also part of the invention. Examples of retinal tissue disorders that may be treated include eye diseases and disorders resulting from sensorineural pathologies, such as loss of sight, which may also be treated using the methods of the invention. These disorders of the eye include, but are not necessarily limited to: various retinopathies, such as hypertensive retinopathy, diabetic retinopathy and occlusive retinopathy; also injuries and disorders resulting in retinal degeneration, such as retinal tearing and detachment and inherited diseases, such as retinitis pigmentosa; also age-related macular degeneration; diseases of the optic nerve; glaucoma and retinal ischemia.
The methods of the invention make use of the fact that the various neuregulin proteins are encoded by the same gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to p185erbB2 (or related receptors erbB3 and erbB4) and activation of the same. Products of this gene are used to show retinal cell survival activity (see Example 2, below). This invention provides a use for all of the known products of the neuregulin gene (described herein and in the references listed above), which have the stated activities as promoting retinal cell function. Most preferably, recombinant human GGF2 (rhGGF2) is used in these methods.
The invention also relates to the use of other, not yet naturally isolated, splicing variants of the neuregulin gene. FIG. 12 shows the known patterns of splicing. These patterns are derived from polymerase chain reaction experiments (on reverse transcribed RNA), analysis of cDNA clones (as presented within) and from analysis of published sequences encoding neuregulins (Peles et al., Cell 69:205, 1992; Wen et al., Cell 69:559, 1992; Wen et al., Mol. Cell Biol. 14:1909, 1994) These patterns, as well as additional patterns disclosed herein, represent probable splicing variants which exist. The splicing variants are fully described in Goodearl et al., U.S. Ser. No. 08/036,555, filed Mar. 24, 1993, incorporated herein by reference.
Advantages of the present invention include the development of new therapeutic approaches to injury or diseases of the eye, more specifically degenerative diseases of the retina, based on the promotion of retinal cell function through the use of neuregulins. Loss of retinal cells is a common feature of degenerative eye diseases, and there are no available treatments, including growth factors , that prevent the death of retinal ganglion cells. The factor can be formulated for intraocular injection and administered to patients that suffer from degenerative disorders, which lead to loss of sight. Thus, this approach to therapy can halt or slow the progressive loss of sight, which ensues in various eye diseases.